Angiogenesis is the generation of new blood vessels in a tissue or organ. Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific, restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation of the corpus luteum, endometrium and placenta.
Capillary blood vessels are composed of endothelial cells and pericytes, surrounded by a basement membrane. Angiogenesis begins with the erosion of the basement membrane by enzymes released from endothelial cells and leukocytes. Endothelial cells, lining the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating a new blood vessel.
Uncontrolled angiogenesis is a hallmark of cancer. In 1971, Dr. Judah Folkman proposed that tumor growth is dependent upon angiogenesis. See, e.g., Folkman, New England Journal of Medicine, 285:1182-86 (1971). According to Dr. Folkman, a tumor can only grow to a certain size without the growth of additional blood vessels to nourish the tumor. In its simplest terms, this proposition states: that “once tumor ‘take’ has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor.” Tumor ‘take’ is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume, and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, pulmonary micro-metastasis in the early prevascular phase in mice would be undetectable except by high power microscopy on histological sections.
As early as 1945, Algire, et al., J. Nat. Cancer Inst., 6:73-85 (1945), showed that the growth rate of tumors implanted in subcutaneous transparent chambers in mice is slow and linear before neovascularization, and rapid and nearly exponential after neovascularization. In 1966, Dr. Folkman reported that tumors grown in isolated perfused organs where blood vessels do not proliferate are limited to 1-2 mm3 but expand rapidly to >1000 times this volume when they are transplanted in mice and become neovascularized. See, e.g, Folkman, et al, Anals of Surgery, 164:491-502 (1966).
Tumor growth in avascular cornea proceeds slowly and at a linear rate, but switches to exponential growth after neovascularization. See, eg., Gimbrone, Jr., et al., J. Nat. Cancer Inst., 52:421-27 (1974)). Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye remain viable, avascular, and limited in size to <1 mm3. Once they are implanted on the iris vascular bed, they become neovascularized and grow rapidly, reaching 16,000 times their original volume within two weeks. See, eg., Gimbrone, Jr. et al., J. Exp. Med., 136:261-76.
When tumors are implanted on the chick embryo chorioallantoic membrane, they grow slowly during an avascular phase of >72 hours, but do not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansion occurs within 24 hours after the onset of neovascularization, and by day 7 the vascularized tumors reach a mean diameter of 8.0+2.5 mm. See, eg., Knighton, British, J. Cancer, 35:347-56 (1977)).
Vascular casts of metastasis in the rabbit liver reveal heterogeneity in size of the metastasis, but show a relatively uniform cut-off point for the size at which vascularization is present. Tumors are generally avascular up to 1 mm in diameter, but are neovascularized beyond that diameter. See, eg., Lien, et al., Surgery, 68:334-40 (1970).
In transgenic mice which develop carcinomas in the beta cells of the pancreatic eyelets, pre-vascular hyperplastic eyelets are limited in size to <1 mm. At 6-7 weeks of age, 4-10% of the eyelets become neovascularized, and from these eyelets arrive large vascularized tumors of more than 1,000 times the volume of the pre-vascular eyelets. See, eg., Folkman, et al., Nature, 339:58-61 (1989).
It has been shown that tumors can be treated by inhibiting angiogenesis rather than inhibiting proliferation of the tumor cells themselves. For example, Kim et al., Nature, 362:841044 (1993), show that a specific antibody against VEGF (vascular endothelial growth factor) reduces micro-vessel density and causes “significant or dramatic” inhibition of growth of three human tumors which rely on VEGF as their sole mediator of angiogenesis (in nude mice). The antibody does not inhibit growth of the tumor cells in vtiro. Further, Hori, et al., Cancer, Resp. 51:6180-84 (1991), shows that anti-bFGF monoclonal antibody causes 70% inhibition of growth of a mouse tumor which is dependent upon secretion of bFGF as its only mediator of angiogenesis. The antibody does not inhibit growth of the tumor cells in vitro. Intraperitoneal injection of bFGF has also been shown to enhance growth of a primary tumor and its metastasis by stimulating growth of capillary endothelial cells in the tumor. The tumor cells themselves lack receptors for bFGF and bFGF is not a mitogen for the tumor cells in vitro. See, e.g., Gross, et al., Proc. Am. Assoc. Cancer Res., 31:79 (1990). A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growth and metastasis in vivo, but is much less active in inhibiting tumor cell proliferation in vitro. It inhibits vascular endothelial cell proliferation half-maximally at 4 logs lower concentration than it inhibits tumor cell proliferation. See, eg., Ingber, et al., Nature, 48:555-57 (1990).
There is also indirect clinical evidence that tumor growth is angiogenesis dependent. For example, human retinoblastomas that are metastatic to the vitreous develop into avascular spiroids which are restricted to <1 mm3 despite the fact that they are viable and incorporate 3H-Thymidine (when removed from an enucleated eye and analyzed in vitro). Carcinoma of the ovary metastasizes to the peritoneal membrane as tiny avascular white seeds (1-3 mm3). These implants rarely grow larger until one or more of them become neovascularized. Intensity of neovascularization in breast cancer (see, e.g., Weidner, et al., New Eng. J. of Med., 324:1-8 (1991); Weidner, et al., J. Nat. Cancer Inst., 84:1875-87 (1992)) and in prostate cancer (Weidner, et al., Am. J. Pathol., 143 (2):401-09 (1993)) correlates highly with risk of future metastasis.
Metastasis from human cutaneous melanoma is rare prior to neovascularization. The onset of neovascularization leads to increased thickness of the lesion and an increased risk of metastasis. See, eg., Srivastava, et al., Am. J. Pathol., 133:419-23 (1988)). In bladder cancer, the urinary level of an angiogenic protein, bFGF is a more sensitive indicator of status and extensive disease than is cytology. See, e.g., Nguyen, et al., J. Nat. Cancer, Inst., 85:241-42 (1993).
Angiogenesis has been associated with a number of different types of cancer, including solid tumors and blood-borne tumors. Solid tumors with which angiogenesis has been associated include, but are not limited to, rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma. Angiogenesis also has been linked with breast cancer, prostate cancer, lung cancer, and colon cancer. Angiogenesis is also associated with blood-borne tumors, such as leukemias, lymphomas, multiple myelomas, and any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver and spleen. It is believed too that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia and lymphoma tumors and multiple myeloma diseases.
One of the most frequent angiogenic diseases of childhood is the hemangioma. A hemangioma is a tumor composed of newly-formed blood vessels. In most cases the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cave and infiltrated forms and create clinical complications. Systemic forms of hemangiomas, hemangiomatoses, which have a high mortality rate. Therapy-resistant hemangiomas exist that can not be treated with therapies currently in use.
Thus, it is clear that angiogenesis plays a major role in the metastasis of cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could overt the damage caused by the invasion of the new micro vascular system. Therapies directed at control of angiogenic processes could lead to the abrogation or mitigation of these diseases.
Several classes of compounds that inhibit angiogenesis are being investigated as therapeutic agents. These are, for example, thalidomide and thalidomide analogs (U.S. Pat. Nos. 6,235,756 (The Children's Medical Center Corporation); 6,420,414 (The Children's Medical Center Corporation); 6,476,052 (Celgene Corporation)); quinolinones (U.S. Pat. No. 6,774,237 (Chiron Corporation)); serine proteases and kallikreins (U.S. Pat. No. 6,544,947 (EntreMed Inc.)); VEGF analogs and antagonists (U.S. Pat. Nos. 6,783,953 (Janssen Pharmaceutica N.V.); 6,777,534 (Children's Medical Center Corporation)); peptides and proteins that bind Angiostatin™ or Endostatin™ (U.S. Pat. No. 6,201,104 (EntreMed Inc.)); cathepsin V-like polypeptides (U.S. Pat. No. 6,783,969 (Nuvelo Inc.)); other antiangiogenic peptides (U.S. Pat. No. 6,774,211 (Abbott Laboratories); 4-anilino-quinazolines (WO 2002/092578, WO 2002/092577, WO 2002/016352, WO 2001/032651, WO 2000/047212 (Astrazeneca)); phthalazines (WO 2004/033042 (Novartis), WO 2003/022282 (Novartis), WO 2002/012227 (Astrazeneca), WO 2001/010859 (Bayer), WO 98/35958 (Novartis)); isothiazoles (WO 99/62890 (Pfizer)); and indolinones (WO 2001/037820, WO 2000/008202, WO 98/50356, WO 96/40116 (Sugen Inc.)).
Several review articles report the use of angiogenesis inhibitors as therapeutic agents. These articles include Mazitschek et al. Current Opinion in Chemical Biology, 8(4): 432-441 (2004); Underiner, et al., Current Medicinal Chemistry, 11(6): 731-745 (2004); Manley, et al., Biochimica et Biophysica Acta, 1697(1-2): 17-27 (2004); Alessi, et al., Biochimica et Biophysica Acta, 1654(1): 39-49 (2004); Tortora, et al., Current Pharmaceutical Design, 10(1): 11-26 (2004).
Uncontrolled cell proliferation is another hallmark of cancer. Cancerous tumor cells typically have some form of damage to the genes that directly or indirectly regulate the cell-division cycle.
Cyclin-dependent kinases (CDKs) are enzymes which are critical to cell cycle control. See,, e.g., Coleman et al., Annual Reports in Medicinal Chemistry, 32: 171-179 (1997). These enzymes regulate the transitions between the different phases of the cell cycle, such as the progression from the G1 phase to the S phase (the period of active DNA synthesis), or the progression from the G2 phase to the M phase, in which active mitosis and cell-division occurs. See, e.g., the articles on this subject appearing in Science, 274: 1643-1677 (6 Dec. 1996).
CDKs are composed of a catalytic CDK subunit and a regulatory cyclin subunit. The cyclin subunit is the key regulator of CDK activity, with each CDK interacting with a specific subset of cyclins: e.g. cyclin A (CDK1, CDK 2). The different kinase/cyclin pairs regulate progression through specific stages of the cell cycle. See, e.g., Coleman, supra.
Aberrations in the cell cycle control system have been implicated in the uncontrolled growth of cancerous cells. See, e.g., Kamb, Trends in Genetics, 11: 136-140 (1995); and Coleman, supra. In addition, changes in the expression of or in the genes encoding CDK's or their regulators have been observed in a number of tumors. See, e.g., Webster, Exp. Opin. Invest. Drugs, 7: 865-887 (1998), and references cited therein. Thus, there is an extensive body of literature validating the use of compounds inhibiting CDKs as anti-proliferative therapeutic agents. See, e.g. U.S. Pat. No. 5,621,082; EP 0 666 270 A2; WO 97/16447; and the references cited in Coleman, supra, in particular reference no. 10. Thus, it is desirable to identify chemical inhibitors of CDK kinase activity.
It is particularly desirable to identify small molecule compounds that may be readily synthesized and are effective in inhibiting one or more CDKs or CDK/cyclin complexes, for treating one or more types of tumors.
Several classes of compounds that inhibit cyclin-dependent kinases have been and are being investigated as therapeutic agents. These are, for example, analogs of Flavopiridol (U.S. Pat. No. 5,733,920 (Mitotix); WO 98/1344 (Bristol-Myers Squibb); WO 97/42949 (Bristol-Meyers Squibb)); purine derivatives (WO 98/05335 (CV Therapeutics); WO 97/20842 (CNRS)); acridones and benzothiadiazines (WO 98/49146 A2 (US Dept. of Health and Human Services)); and antisense (U.S. Pat. No. 5,821,234 (Stanford University)). Furthermore, certain N,N-substituted dihydropyrazolobenzodiazepines have been disclosed in an article discussing CNS-acting compounds. See, M. A. Berghot, Arch. Pharm. 325:285-289 (1992).
There continues to be a need for easily synthesized, small molecule compounds for the treatment of one or more types of tumors, in particular through regulation of angiogenesis and/or CDKs.